Carbohydrates I: Fermentation and beta-Galactosidase Activity

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



O



H



H

OH



H

O



H



O

H

OH



H



H

H



H



OH



OH



CH2OH



CH2OH



CH2OH



CH2OH

HO



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20. Carbohydrates I:

Fermentation

andßGalactosidase

Activity



OH



β-galactosidase



H



HO



H

OH



H



OH

H



H



OH



β-galactose



Lactose

(β-form)



the production of acid and sometimes gas. S. cholerae-suis

is ȋ-galactosidase positive. The yeast, Saccharomyces cerevisiae, will ferment glucose (but not sucrose) to produce

gas, but no acid. S. cerevisiae is ȋ-galactosidase negative.



OH



H

OH



H



H



+



H



H

H2O



O



O



OH



HO



H



β-glucose



ȋ-D-galactopyranoside), can be used. ȋ-galactosidase

catalyzes the hydrolysis of ONPG as follows:

ONPG + H2O



ȋ-galactosidase



galactose + o-nitrophenol.



Principles

Fermentations are energy-producing biochemical reactions in which organic molecules serve both as electron

acceptors and donors. The ability of microorganisms to

ferment carbohydrates and the types of products formed

are very useful in identification. A given carbohydrate

may be fermented to a number of different end products

depending upon the microorganism involved (figure

20.1). These end products (alcohols, acids, gases, or

other organic molecules) are characteristic of the particular microorganisms. For example, if fermenting bacteria are grown in a liquid culture medium containing the

carbohydrate glucose, they may produce organic acids

as by-products of the fermentation. These acids are released into the medium and lower its pH. If a pH indicator such as phenol red or bromcresol purple is included

in the medium, the acid production will change the

medium from its original color to yellow (figures 20.2,

20.3; see also appendix E).

Gases produced during the fermentation process

can be detected by using a small, inverted tube, called

a Durham tube (named after Herbert Edward

Durham, English bacteriologist, 1866–1945), within

the liquid culture medium. After adding the proper

amount of broth, Durham tubes are inserted into each

culture tube. During autoclaving, the air is expelled

from the Durham tubes, and they become filled with

the medium. If gas is produced, the liquid medium inside the Durham tube will be displaced, entrapping

the gas in the form of a bubble (figure 20.2).

Some microorganisms, such as E. coli, can use lactose as their sole source of carbon. An essential enzyme

in the metabolism of this sugar is ȋ-galactosidase.

ȋ-galactosidase hydrolyzes lactose to galactose and

glucose as shown in a previous diagram.

Instead of lactose, the natural substrate of this enzyme, an artificial substrate, ONPG (o-nitro-phenyl-



ONPG is colorless but upon hydrolysis yields

o-nitrophenol, which is yellow in an alkaline solution. If an ONPG disk or KEY tablet is incubated

with a bacterial culture and the culture turns yellow,

this is the positive test for ȋ-galactosidase activity

(figure 20.4).

Each of the above tests is important in the identification of certain bacteria.



Procedure: Durham Tube

First Period

1. Label five of the specified culture tubes with your

name, date, and type of culture medium.

2. Label the first tube E. coli; the second, S.

typhimurium; the third, A. faecalis; the fourth, S.

cerevisiae; and the fifth, “control.”

3. Using aseptic technique (see figure 14.3), inoculate

each tube with the corresponding microbial culture.

Leave the fifth tube uninoculated. Care should be

taken during this step not to tip the fermentation

tube, as this may accidentally force a bubble of air

into the Durham tube and give a false-positive

result. The tubes may be mixed by rolling them

back and forth between the palms of the hands.

4. Place the five tubes in a test-tube rack and

incubate at 35°C for 24 to 48 hours.



Second Period

1. Ideally, the tubes should be examined carefully

between 2 to 4 hours, at 8 hours, and 18 hours in

order to avoid false negatives due to reversal of

the fermentation reactions that may occur with

long incubations.

2. Examine all carbohydrate broth cultures for

evidence of acid (A), or acid and gas (A/G)

production. Use figure 20.2 as a reference. Acid



Carbohydrates I: Fermentation and ȋ-Galactosidase Activity



127



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Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



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20. Carbohydrates I:

Fermentation

andßGalactosidase

Activity



Figure 20.1 Outline of Major Fermentation Pathways. Microorganisms produce various waste products when they ferment glucose.

The by-products released (shaded boxes) are often characteristic of the microorganisms and can be used as identification tools.

Sugars

Glycolysis

1



2



CO2



NADH



Lactic

acid



Ethanol

+

CO2



NADH

Pyruvate



Acetaldehyde



CO2



CO2



CoASH



3



4

α-acetolactate



Oxaloacetate

NADH



CO2



Formic acid



Acetyl-CoA

Malate

5



ADP



Acetoin

CO2



H2



H 2O



ATP



NADH



Ethanol



Fumarate

6



Acetate



Acetoacetyl-CoA



2,3,-butanediol

NADH



Succinate



CO2



Acetone

CO2



NADH

Propionic

acid



Butyryl-CoA

NADH



Isopropanol



Butanol



Butyric acid



1. Lactic acid fermentation. Lactic acid bacteria (Streptococcus, Lactobacillus).

2. Alcoholic fermentation. Zymomonas, Saccharomyces.

3. Propionic acid fermentation. Propionic acid bacteria (Propionibacterium).

4. 2,3,-butanediol fermentation. Enterobacter, Serratia, Bacillus.

5. Mixed acid fermentation. Enteric bacteria (Escherichia, Enterobacter, Salmonella, Proteus).

6. Butyric acid fermentation. Clostridium.



Figure 20.2 Carbohydrate Fermentation. (a) Possible carbohydrate fermentation patterns of microorganisms, with phenol red as the

pH indicator. (b) The tube on the left is the control. The next tube shows alcohol fermentation. Notice the gas bubble at the top. The third

tube from the left shows no carbohydrate fermentation (negative). The tube on the right shows acid and gas production.



Durham

tube



Gas

(bubble)



Acid

(yellow)



Original

color

(red)



Original

color

(red)



(b)

No

fermentation



Acid

production



Acid and

gas production



(a)



128



Biochemical Activities of Bacteria



Alcohol

fermentation



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



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20. Carbohydrates I:

Fermentation

andßGalactosidase

Activity



Figure 20.3 Carbohydrate Fermentation Using KEY Rapid

Fermentation Tablets. The color change from red (right tube) to

yellow (left tube) indicates acid production. Fermentation may be

apparent within 30 minutes and usually is complete in 6 hours, but

tubes should be held for 48 hours before being discarded. Gas

production is shown by the appearance of bubbles near the bottom

of the tube. Both of these tubes are negative for gas production.



production is detected by the medium turning

yellow, and gas production by a gas bubble in the

Durham tube.

3. The control tube should be negative for acid and

gas production, and should have no turbidity.

4. Based on your observations, determine and record

in the report for exercise 20 whether or not each

microorganism was capable of fermenting the

carbohydrate substrate with the production of

acid, or acid and gas. Compare your results with

other students who used other sugars.



Procedure: Sugar-Differentiation Disk

First Period

1. Label five tryptic agar base tubes with your name,

date, and the sugar to be studied.

2. Label the first tube E. coli; the second,

S. cholerae-suis; the third, A. faecalis; the fourth,

S. cerevisiae; and the fifth, “control.”

3. Using sterile forceps (or forceps that have been

dipped in 70% ethanol and flamed), aseptically

add one of your assigned sugar disks to each tube.

4. Inoculate each tube except the control in the

following way. Heavily load the inoculating

needle with microbial paste, then stab the deep

until the needle reaches about d to i of the agar

depth. The inoculating needle should strike the



Figure 20.4 ONPG Test Using KEY Tablets. An ONPG

positive reaction can be detected within 30 minutes by the

appearance of a yellow color (tube on the left). A positive ONPG

test shows that the bacteria being tested contain the enzyme betagalactosidase, can ferment lactose, and may be properly classed as

lactose fermenters. The tube on the right has not changed color;

thus the bacteria would be ONPG negative.



sugar-differentiation disk off-center and push it

into the soft agar. The control tube should be

stabbed with a sterile inoculating needle.

5. Incubate the tubes at 35°C for 24 hours.



Second Period

1. Examine the tubes after 2 to 4 hours, about 8

hours, and 18 hours.

2. Acid production is shown by yellowing of the

medium around the disk, which often spreads

throughout the agar. Gas production yields

bubbles and splitting of the soft agar. Remember

that positive-acid results can reverse with

further incubation. Therefore, yellowing of the

agar at 2 to 4 hours is a positive result, even if

the tube turns red to violet upon longer

incubation.

3. Compare your results with those of students who

used different sugars.



ONPG Disks or Tablets

1. Dispense 0.2 ml of sterile 0.85% NaCl (Difco

disks) or 1 ml (about 40 drops with a Pasteur

pipette) of distilled water (KEY tablets) into four

sterile test tubes.

2. Label each tube with the microorganism to be

inoculated. Add your name and the date.

3. Suspend a loopful of paste (concentrated

microorganisms) into each tube.



Carbohydrates I: Fermentation and ȋ-Galactosidase Activity



129



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



20. Carbohydrates I:

Fermentation

andßGalactosidase

Activity



4. Place an ONPG disk or tablet into each tube and

incubate at 35°C.

5. Check at 20 minutes and 4 hours (the KEY tablet

test can run up to 6 hours).

6. A positive ȋ-galactosidase test is indicated by a

yellow color; no color change indicates a negative

test.

7. Record your results in the report for exercise 20.



130



Biochemical Activities of Bacteria



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HINTS AND PRECAUTIONS

(1) The amount of inoculum placed in each tube should

be small. Too much inoculum may lead to overgrowth

and neutralization of acidic fermentation products by

the bacteria, thus resulting in false negatives. (2) Do not

vortex the fermentation tubes when inoculating because

this can force air into the Durham tubes and result in

false-positive recordings for gas production.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



20



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20. Carbohydrates I:

Fermentation

andßGalactosidase

Activity



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Carbohydrates I: Fermentation and β-Galactosidase Activity

1. Complete the following table on carbohydrate fermentation.

Biochemical Results



Bacteria

E. coli



S. cholerae-suis



A. faecalis



S. cerevisae



Lactose

(A), (A/G), (–)



_______________



_______________



_______________



_______________



Dextrose

(A), (A/G), (–)



_______________



_______________



_______________



_______________



Sucrose

(A), (A/G), (–)



_______________



_______________



_______________



_______________



A = Acid production.

A/G = Acid and gas production.

G = Gas production.

– = No growth or no change (alkaline).



2. Indicate whether the following bacteria have β-galactosidase activity.

a. A. faecalis ___________________________________________________________________________

b. E. coli ______________________________________________________________________________

c. S. cholerae-suis ______________________________________________________________________

d. S. cerevisiae _________________________________________________________________________



131



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Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



20. Carbohydrates I:

Fermentation

andßGalactosidase

Activity



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Review Questions

1. Define fermentation.



2. Do all microorganisms produce the same end product from pyruvate? Explain your answer.



3. What is the purpose of the phenol red or bromcresol purple in the fermentation tube?



4. What is the function of the Durham tube in the fermentation tube?



5. What are some of the metabolic end products produced by the different microorganisms used in this

experiment?



6. What is the color of phenol red at an acid pH?



7. What is the function of β-galactosidase?



132



Biochemical Activities of Bacteria



Harley−Prescott:

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IV. Biochemical Activities

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21. Carbohydrates II: Triple

Sugar Iron Agar Test



E X E RC I S E



21



Carbohydrates II: Triple Sugar Iron Agar Test

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame. Be careful

when working with these bacteria, especially Shigella

dysenteriae, as they are known pathogens. Keep all culture tubes upright in a test-tube rack or in empty cans.



Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jinOH-sah)

Shigella flexneri (shi-GEL-la flex-ner-i)



Why Are the Above Bacteria Used

in This Exercise?



Materials per Student

24- to 48-hour tryptic soy broth cultures of

Alcaligenes faecalis (ATCC 8750), Escherichia

coli (ATCC 11229), Proteus vulgaris (ATCC

13315), Pseudomonas aeruginosa (ATCC

10145), and Shigella flexneri (ATCC 12661)

5 triple sugar iron agar slants

Bunsen burner

inoculating needle

incubator set at 35°C

test-tube rack



Learning Objectives

Each student should be able to

1. Understand the biochemical reactions involved in

the triple sugar iron agar test

2. Differentiate among members of the family

Enterobacteriaceae

3. Distinguish between the Enterobacteriaceae and

other intestinal bacteria

4. Perform a TSI test



Suggested Reading in Textbook

1. Carbohydrate catabolism, section 9.7.

2. The Enterobacteriaceae, section 22.3.



Pronunciation Guide

Alcaligenes faecalis (al-kah-LIJ-e-neez fee-KAL-iss)

Escherichia coli (esh-er-I-ke-a KOH-lee)

Proteus vulgaris (PRO-tee-us vul-GA-ris)



This exercise will provide the student experience in using the

triple sugar iron agar test to differentiate among the members

of the family Enterobacteriaceae and between Enterobacteriaceae and other intestinal bacteria. The authors have chosen three common bacteria in the family Enterobacteriaceae:

Escherichia coli, Proteus vulgaris, and Shigella flexneri. All

three are facultatively anaerobic gram-negative rods. In a

TSI tube, E. coli produces an acid butt, an acid or alkaline

slant, and no H2S, but does produce gas. P. vulgaris produces an acid butt, an acid or alkaline slant, H2S, and gas. S.

flexneri produces an acid butt, an alkaline slant, no H2S, and

no gas. For the other intestinal bacteria, the authors have

chosen Alcaligenes faecalis and Pseudomonas aeruginosa.

Both of these intestinal bacteria are gram-negative aerobic

rods. In a TSI tube, A. faecalis produces an alkaline butt, alkaline slant, H2S, and gas; P. aeruginosa, an acid butt, alkaline slant, H2S, and gas.



Principles

As originally described in 1911 by F. F. Russell, the

triple sugar iron (TSI) agar test is generally used for

the identification of enteric bacteria (Enterobacteriaceae). It is also used to distinguish the Enterobacteriaceae from other gram-negative intestinal bacilli by

their ability to catabolize glucose, lactose, or sucrose,

and to liberate sulfides from ferrous ammonium sulfate

or sodium thiosulfate. (See exercise 24 for the biochemistry of H2S production.) TSI agar slants contain a 1%

concentration of lactose and sucrose, and a 0.1% glucose concentration. The pH indicator, phenol red, is also



133



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Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Figure 21.1 Triple Sugar Iron Reactions (TSI-1) and Their

Interpretation. (a) The tube on the left has a yellow

butt (acid), red slant (alkaline), H2S production as indicated by

blackening of the agar, and no gas production. (b) The tube on the

right shows no H2S formation, a yellow slant (acid), gas production,

and an acid butt. Note that the gas on the bottom has lifted the agar.



(a)



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21. Carbohydrates II: Triple

Sugar Iron Agar Test



Figure 21.2 Triple Sugar Iron Reactions (TSI-2) and

Their Interpretation. (a) The tube on the left has a red butt

(alkaline), red slant (alkaline), and no acid or H2S production.

(b) The tube on the right has a yellow slant (acid), yellow butt

(acid), and no gas or H2S production.



Tube a



Tube b



Slant



K



A



Butt



A



A



Gas



–



H 2S



+



(b)



(a)



(b)



Tube a



Tube b



Slant



K



A



Butt



K



A



+



Gas



–



–



–



H 2S



–



–



incorporated into the medium to detect acid production

from carbohydrate fermentation (see exercise 20).

Often Kligler Iron Agar (named after I. J. Kligler

in 1917), a differential medium similar to TSI, is used

to obtain approximately the same information.

TSI slants are inoculated by streaking the slant

surface using a zig-zag streak pattern and then stabbing the agar deep with a straight inoculating needle

(see figure 14.5). Incubation is for 18 to 24 hours in

order to detect the presence of sugar fermentation, gas

production, and H2S production. The following reactions may occur in the TSI tube (figures 21.1–21.3):

1. Yellow butt (A) and red slant (A) due to the

fermentation of glucose (phenol red indicator turns

yellow due to the persisting acid formation in the

butt). The slant remains red (alkaline) (K) because

of the limited glucose in the medium and, therefore,

limited acid formation, which does not persist.

2. A yellow butt (A) and slant (A) due to the

fermentation of lactose and/or sucrose (yellow slant

and butt due to the high concentration of these

sugars) leading to excessive acid formation in the

entire medium.



3. Gas formation noted by splitting of the agar.

4. Gas formation (H2S) seen by blackening of the

agar.

5. Red butt (K) and slant (K) indicates that none of

the sugars were fermented and neither gas nor

H2S were produced.

Table 21.1 gives reactions usually expected from

some of the more frequently encountered genera of

the Enterobacteriaceae. Figure 21.4 summarizes the



Table



21.1 Results of TSI Reaction

TSI Reaction



Bacterium



Butt



Slant



H2S



Gas



Enterobacter

Escherichia

Klebsiella

Citrobacter

Proteus vulgaris

Edwardsiella

Morganella

Serratia

Shigella

Salmonella typhi



A

A

A

A

A

A

A

A

A

A



A

A or K

A

K or A

A or K

K

K

K or A

K

K



–

–

–

V

+

V

–

–

–

+



+

+

+

+

+

+

+

V

–

–



A = acid, K = alkaline, V = varies between species



134



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



21. Carbohydrates II: Triple

Sugar Iron Agar Test



Figure 21.3 Triple Sugar Iron Reactions (TSI-3) and

Their Interpretation. (a) The tube on the left is an uninoculated

control. Notice the red color. (b) The second tube from the left

has a yellow slant (acid), yellow butt (acid), gas production at the

bottom of the tube, and no H2S production. This would indicate a

weak lactose fermenter. (c) The third tube from the left has a red

slant (alkaline), red butt (alkaline), and the black indicates H2S

production, but no gas. (d) The tube on the right has a red slant

(alkaline), yellow butt (acid), H2S production, but no gas

production. This would indicate a nonlactose fermenter.



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possible reactions and results in TSI for the various

bacteria used in this experiment.



Procedure

First Period

1. Label each of the TSI agar slants with the name of

the bacterium to be inoculated. Use one of the tubes

as a control. Place your name and date on each tube.

2. Using aseptic technique (see figure 14.3), streak

the slant with the appropriate bacterium and then

stab the butt. Screw the caps on the tubes but do

not tighten!

3. Incubate for only 18 to 24 hours at 35°C for

changes in the butt and on the slant. Tubes should

be incubated and checked daily for up to seven

days in order to observe blackening.



Second Period



(a)



(c)



(b)



1. Examine all slant cultures for the color of the

slant and butt, and for the presence or absence of

blackening within the medium.

2. Record your results in the report for exercise 21.



(d)



Tube a



Tube b



Tube c



Tube d



Slant



–



A



K



K



Butt



–



A



K



A



Gas



–



+



–



–



H 2S



–



–



+



+



HINTS AND PRECAUTIONS

(1) If screw-cap tubes are used, leave the caps loose about

b turn after inoculating the tubes to prevent excessive disruption of the agar should large amounts of gas be produced during incubation. (2) Record the butt as acid production if the black color of FeS masks the color in the butt.



Figure 21.4 The Possible Reactions and Results in TSI Agar for the Various Bacteria Used in This Experiment.



No carbohydrate fermentation or hydrogen sulfide production

Example: Alcaligenes faecalis

glucose, lactose, sucrose ¡ glucose, lactose, sucrose

(red slant/red butt)

(K; red slant/red butt)

cysteine ¡ cysteine

(no black color)

Glucose fermentation only

Example: Shigella flexneri

glucose ¡ decrease in pH due to acid

(red butt) (A; yellow butt)

Carbohydrates II: Triple Sugar Iron Agar Test



(continued)



135



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Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



21. Carbohydrates II: Triple

Sugar Iron Agar Test



© The McGraw−Hill

Companies, 2002



Figure 21.2 (continued)



lactose, sucrose ¡ lactose, sucrose

(red slant)

(K; red slant)

cysteine ¡ cysteine

(no black color)

Glucose fermentation only with hydrogen sulfide production

Example: Pseudomonas aeruginosa

glucose ¡ decrease in pH due to acid

(red butt)

(A; yellow butt)

lactose, sucrose ¡ lactose, sucrose

(red slant)

(K; red slant)

cysteine ¡ H2S production

H2S ϩ FeSO4 ¡ FeS

(black color in media)

Lactose and/or sucrose and glucose fermentation

Example: Escherichia coli

glucose ¡ decrease in pH due to acid

(red butt)

(A; yellow butt)

lactose and/or sucrose ¡ decrease in pH due to acid

(red butt)

(A; yellow slant)

cysteine ¡ cysteine

(no black color in media)

Lactose and/or sucrose and glucose fermentation with hydrogen sulfide (H2S) production

Example: Proteus vulgaris

glucose ¡ decrease in pH due to acid

(red butt)

(A; yellow butt)

lactose and/or sucrose ¡ decrease in pH due to acid

(red slant)

(A; yellow butt)

cysteine ¡ H2S production



136



H2Sϩ FeSO4 ¡ FeS production

(black color in media)



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



21



Laboratory Report



© The McGraw−Hill

Companies, 2002



21. Carbohydrates II: Triple

Sugar Iron Agar Test



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Carbohydrates II: Triple Sugar Iron Agar Test

1. Complete the following table on the TSI test.

Carbohydrate Fermentation

Bacterium



Butt Color



Slant Color



H2S Production

Black



H2S



A. faecalis



______________



__________________



____________



____________



E. coli



______________



__________________



____________



____________



P. vulgaris



______________



__________________



____________



____________



P. aeruginosa



______________



__________________



____________



____________



S. flexneri



______________



__________________



____________



____________



137